By combining innovative, cutting edge technologies with established approaches, the proposed research will uncover fundamental mechanistic principles governing clathrin-mediated endocytosis (CME) in mammalian cells. Expression of fluorescent protein fusions at endogenous levels in genome-edited cells will allow more faithful reporting of endocytic dynamics than could have previously been achieved. As a result, effects of RNAi, physical and small molecule perturbations will be more sensitively detected and more powerfully analyzed than was previously possible. The expected outcome of this research is an understanding of how coordinated activities of dozens of proteins are harnessed for the mechanochemical process of endocytic vesicle formation. Because multiple proteins will be analyzed, holistic design principles for the endocytic system will be revealed. Three aims will be addressed: 1. Spatio-temporal dynamics of endocytic protein recruitment and vesicle formation: Using genome-edited, stable cell lines expressing pair-wise combinations of five different endocytic protein-fluorescent protein fusions at native levels, real-time imaging and analytical software will be used to determine precise recruitment profiles, providing powerful insights into function, mechanism, regulation and system logic. The data will be modeled mathematically and will generate hypotheses for functional studies. Mathematical modeling will also explore the hypothesis that lipids play an active role in generation of membrane-bending and scission forces. 2. Elucidation of endocytic protein functions in vivo: Real-time imaging of genome-edited cell lines will sensitively test the impact of function perturbations on CME. Functions of endocytic proteins in their biological context will be elucidated using RNAi and small molecule inhibitors. Functions of known endocytic proteins and three novel endocytic proteins identified in a bioinformatic screen will be tested. The ultrastructural underpinnings of real-time observations will be revealed by electron microscopy. Chemical-genetic strategies will be improved by genome editing to elucidate clathrin light chain function in vivo. 3. Impact of cargo, physical and developmental parameters on endocytic dynamics: The hypotheses that endocytic cargo load and membrane tension affect CME dynamics will be tested. Using genome-edited cell lines of varied tissue origin and stem cells, the hypothesis that CME is fine-tuned and modified developmentally for distinct physiological states will be tested.